Protein purification using immobilized metal affinity chromatography for complement receptor proteins

ABSTRACT

This invention relates to the application of immobilized metal affinity chromatography to the purification of complement receptor proteins.

This application is a 371 of PCT94/07555 filed Jul. 6, 1994 which is acontinuation of 08/09351 filed Jul. 9, 1993, now abandoned.

FIELD OF THE INVENTION

This invention relates to the field of protein purification. Morespecifically, this invention relates to the application of immobilizedmetal affinity chromatography to the purification of complement receptorproteins.

BACKGROUND OF THE INVENTION

Historically, protein purification schemes have been predicated ondifferences in the molecular properties of size, charge and solubilitybetween the protein to be purified and undesired protein contaminants.Protocols based on these parameters include size exclusionchromatography, ion exchange chromatography, differential precipitationand the like.

Size exclusion chromatography, otherwise known as gel filtration or gelpermeation chromatography, relies on the penetration of macromoleculesin a mobile phase into the pores of stationary phase particles.Differential penetration is a function of the hydrodynamic volume of theparticles. Accordingly, under ideal conditions the larger molecules areexcluded from the interior of the particles while the smaller moleculesare accessible to this volume and the order of elution can be predictedby the size of the protein because a linear relationship exists betweenelution volume and the log of the molecular weight. Size exclusionchromatographic supports based on cross-linked dextrans e.g. SEPHADEX®,spherical agarose beads e.g. SEPHAROSE® (both commercially availablefrom Pharmacia AB. Uppsala, Sweden), based on cross-linkedpolyacrylamides e.g. BIO-GEL® (commercially available from BioRadLaboratories, Richmond, Calif.) or based on ethylene glycol-methacrylatecopolymer e.g. TOYOPEARL HW65S (commercially available from ToyoSodaCo., Tokyo, Japan) are useful in the practice of this invention.

Precipitation methods are predicated on the fact that in crude mixturesof proteins the solubilities of individual proteins are likely to varywidely. Although the solubility of a protein in an aqueous mediumdepends on a variety of factors, for purposes of this discussion it canbe said generally that a protein will be soluble if its interaction withthe solvent is stronger than its interaction with protein molecules ofthe same or similar kind. Without wishing to be bound by any particularmechanistic theory describing precipitation phenomena, it is nonethelessbelieved that the interaction between a protein and water moleculesoccurs by hydrogen bonding with several types of uncharged groups, andelectrostatically as dipoles with charged groups, and that precipitantssuch as salts of monovalent cations (e.g., ammonium sulfate) competewith proteins for water molecules, thus at high salt concentrations, theproteins become "dehydrated" reducing their interaction with the aqueousenvironment and increasing the aggregation with like or similar proteinsresulting in precipitation from the medium.

Ion exchange chromatography involves the interaction of chargedfunctional groups in the sample with ionic functional groups of oppositecharge on an adsorbent surface. Two general types of interaction areknown. Anionic exchange chromatography mediated by negatively chargedamino acid side chains (e.g. aspartic acid and glutamic acid)interacting with positively charged surfaces and cationic exchangechromatography mediated by positively charged amino acid residues (e.g.lysine and arginine) interacting with negatively charged surfaces.

More recently affinity chromatography and hydrophobic interactionchromatography techniques have been developed to supplement the moretraditional size exclusion and ion exchange chromatographic protocols.Affinity chromatography relies on the interaction of the protein with animmobilized ligand. The ligand can be specific for the particularprotein of interest in which case the ligand is a substrate, substrateanalog, inhibitor or antibody. Alternatively, the ligand may be able toreact with a number of proteins. Such general ligands as adenosinemonophosphate, adenosine diphosphate, nicotine adenine dinucleotide orcertain dyes may be employed to recover a particular class of proteins.One of the least biospecific of the affinity chromatographic approachesis immobilized metal affinity chromatography (IMAC), also referred to asmetal chelate chromatography. IMAC introduced by Porath et al.(Nature258:598-99(1975) involves chelating a metal to a solid support and thenforming a complex with electron donor amino acid residues on the surfaceof a protein to be separated.

Hydrophobic interaction chromatography was first developed following theobservation that proteins could be retained on affinity gels whichcomprised hydrocarbon spacer arms but lacked the affinity ligand.Although in this field the term hydrophobic chromatography is sometimesused, the term hydrophobic interaction chromatography (HIC) is preferredbecause it is the interaction between the solute and the gel that ishydrophobic not the chromatographic procedure. Hydrophobic interactionsare strongest at high ionic strength, therefore, this form of separationis conveniently performed following salt precipitations or ion exchangeprocedures. Elution from HIC supports can be effected by alterations insolvent, pH, ionic strength, or by the addition of chaotropic agents ororganic modifiers, such as ethylene glycol. A description of the generalprinciples of hydrophobic interaction chromatography can be found inU.S. Pat. No. 3,917,527 and in U.S. Pat. No. 4,000,098. The applicationof HIC to the purification of specific proteins is exemplified byreference to the following disclosures: human growth hormone (U.S. Pat.No. 4,332,717), toxin conjugates (U.S. Pat. No. 4,771,128),antihemolytic factor (U.S. Pat. No. 4,743,680), tumor necrosis factor(U.S. Pat. No. 4,894,439), interleukin-2 (U.S. Pat. No. 4,908,434),human lymphotoxin (U.S. Pat. No. 4,920,196) and lysozyme species(Fausnaugh, J. L. and F. E. Regnier, J. Chromatog. 359:131-146 (1986)).

This invention relates to the application of a combination of ionexchange, IMAC, HIC and size exclusion chromatography to thepurification of complement receptor molecules and complementreceptor-like molecules.

BRIEF DESCRIPTION OF THE INVENTION

This invention relates to a method for purifying a complement receptorprotein from a mixture containing same comprising sequentiallycontacting said mixture with a cationic chromatographic support, metalaffinity chromatographic support, a size exclusion chromatographicsupport and selectively eluting the protein from each support.

In another aspect the invention provides for the purification of acomplement receptor protein from conditioned cell culture mediumcontaining same comprising sequentially subjecting the medium to (a) afirst cationic exchange chromatography, (b) immobilized metal affinitychromatography, (c) hydrophobic interaction chromatography, (d) anionicexchange chromatography, and (e) size exclusion chromatography.

In another aspect this invention provides a method for purifying acomplement receptor protein from a conditioned cell medium comprising:

(a) concentrating the conditioned cell medium;

(b) adsorbing the complement receptor protein onto a cationicchromatographic support;

(c) washing the adsorbed protein with at least one buffer;

(d) eluting the washed protein onto an imnmobilized metal affinitychromatographic support;

(e) adsorbing the eluted protein from step (d);

(f) washing the adsorbed protein with at least one buffer;

(g) eluting the washed protein;

(h) adsorbing the eluted protein from step (g) onto a hyrophobicinteraction chromatographic support;

(i) selectively eluting the protein;

(j) adsorbing the eluate from step (i) onto an anionic exchange support;

(k) eluting the adsorbed protein;

(l) subjecting the eluate from step (k) to size exclusion chromatographyand

(m) recovering the protein therefrom.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to protein purification techniques which haveapplication to the large scale purification of complement receptorproteins. The invention is particularly useful because it permits therecovery of receptor protein of >95% protein purity. The invention maybe applied to the purification of a number of complement receptorproteins and complement receptor-like proteins.

Complement is a group of serum proteins, sequentially activated bylimited proteolysis, that are important effectors of humoral immunity.Activation of complement occurs by interaction of early actingcomplement components with antigen/antibody complexes. Proteolyticfragments resulting from this activation alone or with other proteinsactivate additional complement proteins resulting in a proteolyticcascade reminiscent of the functioning of blood clotting factors.Alternatively, complement can be activated by bacterial cell wallcomponents, proteolytic enzymes (e.g. plasmin) or complex carbohydrates(e.g. inulin). A number of biological activities are mediated bycomponents of the complement system (e.g. immune cytolysis,anaphylatoxin production, bacteriolysis, chemotaxsis, hemolysis,opsonization, and phagocytosis).

Four classes of complement receptors (CR) are known (CR1-CR4).Complement receptor 1 (CR1) is a receptor for complement components C3band C4b. Complement receptor 2 (CR2) is a receptor for component C3dg orC3d. Complement receptor 3 (CR3) is a receptor for C3bi. Complementreceptor 4 (CR4) is a receptor for C3dg.

Complement receptor type 1 (CR1) is present on the membranes oferythrocytes, monocytes/macrophages, granulocytes, B cells, some Tcells, splenic follicular dendritic cells, and glomerular podocytes. CR1binds C3b and C4b and is referred to as the C3b/C4b receptor. Itsprimary sequence has been determined (Klickstein et al., J. Exp. Med.165: 1095-1112 (1987), Klickstein et al., J. Exp. Med. 168:1699-(1988);Hourcade et al., J. Exp Med. 168:1255-1270 (1988)). It is composed of 30short concensus repeats (SCRs) that contain 60-70 amino acids, of which29 of the average 65 amino acids per SCR are conserved. It is proposedthat each SCR forms a three dimensional triple loop structure toughdisulfide linkages with the third and first and the fourth and secondhalf-cystines in disulfide bonds. The SCRs are further organized into 4long homologous repeats (LHRs) of 7 SCRs each. Following a leadersequence, the molecule consists of the most N-terminal LHR-A comprisinga C4b binding domain, the next two repeats, LHR-B and LHR-C comprisingC3b binding domains, and the most C terminal LHR-D followed by 2additional SCRs, a 25 residue putative transmembrane region and a 43residue cytoplasmic tail.

CR1 is a member of a superfamily characterized by SCR homology. Thissuperfamily contains members that also have a C3/C4 binding function,such as CR2, C4bp, factor H, factor B, and C2, as well as proteinswithout this function, such as interleukin-2 receptor, b2-glycoproteinI, C1r, haptoglobin a chain, and factor XIIIb.

CR1 is known to be a glycoprotein and its deduced amino acid sequencehas 24 potential sites for N-linked oligosaccharides in theextracellular region. However, the synthesis of CR1 in the presence oftunicamycin (Lublin et al., J. Biol. Chem. 261: 5736 (1986)) andanalysis of glucosamine content (Sim, Biochem J. 232: 883 (1985)) hassuggested that only 6-8 of the available sites are actually linked tooligosaccharides. The N-terminus of the glycoprotein appears to beblocked.

Four different CR1 allotypes exist that differ in size by 30-50 kDincrements. The gene frequencies of these allelic polymorphisms(allotypes) differ in the human population (Holer et al., Proc. Natl.Acad. Sci. U.S.A. 84:2459-2463 (1987)). The F (or A) allotype iscomposed of 4 LHRs and is about 250 kD; the larger S (or B) allotypecontains a fifth LHR that is a chimera of the 5' half of LHR-B and the3' half of LHR-A and is predicted to have a third C3b binding site (Wonget al., J. Exp. Med. 169: 847 (1989)), and is about 290 kD. The smallestF' (or C) allotype has increased incidence in patients with systemiclupus erythematosis (SLE) (Van Dyne et al., Clin. Exp. Immunol. 68:570(1987) and Dykman et al., Proc. Natl. Acad Sci. USA 80: 1698 (1983)) andmost likely arises from the deletion of LHR-B and one C3b binding site.

A naturally occurring soluble form of CR1 has been detected in theplasma of normal individuals and certain individuals with SLE (Yoon &Fearon J. Immunol. 134: 3332-3338 (1985)). Its characteristics aresimilar to those of erythocyte (cell-surface) CR1 both structurally andfunctionally.

Hourcade et al. (J. Exp. Med. 168: 1255-1270 (1998)) also observed analternative polyadenylation site in the human CR1 transcriptional unitthat was predicted to produce a secreted form of CR1. The mRNA thatarises from this truncated sequence comprises the first 8.5 SCRs of CR1;e.g., the C4b binding domain, and could encode a protein of about 80 kD.When a cDNA corresponding to this truncated sequence was transfectedinto COS cells and expressed, it demonstrated the expected C4b, but notC3b binding activity (Kyrch et al., F.A.S.E.B J. 3:A368 (1989)). Krychet al. also observed a mRNA similar to the predicted one in severalhuman cell lines and postulated that such a truncated soluble form ofCR1 that is able to bind C4b may be synthesized in man.

Several soluble fragments of CR1 have also been generated viarecombinant DNA procedures by eliminating the transmembrane region fromthe DNAs being expressed (Fearon et al., Intl. Patent Publication NumberW089/09220, published Oct. 5, 1989 and Fearon et al. Intl. PatentPublication WO091/05047 published Apr. 18, 1991). The soluble CR1fragments were functionally active, since they were able to bind C3band/or C4b and demonstrate factor I cofactor activity depending upon theregions they contained. In addition they were able to act as inhibitorsof in vitro CR1 functions such as neutrophil oxidative burst, complementmediated hemolysis, and C3a and C5a production. A soluble CR1 construct,encoded by plasmid sCR1/pBSCR1c, also demonstrated in vivo activity in areversed passive arthus reaction (Fearon et al. 1989 & 1991 and Yeh etal., J. Immunol (1991)) and suppressed post-ischemic myocardialinflammation and necrosis (Fearon et al. 1989 & 1990 and Weisman et al.,Science 249: 146-151 (1990)). Furthermore, co-formulation of thesCR1/pBSCR1c product with p-anisoylated humanplasminogen-streptokinase-activator complex (APSAC resulted in similarantihemolytic activity as APSAC alone, indicating that the combinationof the complement inhibitor, sCR1, with a thrombolytic agent, could be auseful combination therapy (Fearon et al., Intl. Patent PublicationNumber WO091/05047 published Apr. 18, 1991).

Complement receptor-like proteins are proteins which may be purified bythe protocol described herein, such protocol being modified if necessaryby routine, non-inventive adjustments that do not entail undueexperimentation. Such proteins include allotypes and alleles of CRs,truncated forms, chemically modified forms such as by PEG treatment, andfusion proteins containing a CR moiety. These proteins are referred toas complement receptor-like because they possess or retain sufficient CRprotein properties to admit to purification by the process of thisinvention. Unless specifically identified otherwise, term complementreceptor protein also includes complement receptor-like proteins.CR-1-like proteins represent a subset of CR-like proteins includingalleles, truncates, chemically modified and fusion proteins derived fromthe CR-1 allotype. Soluble complement receptor 1 (sCR1), defined hereinas a soluble form of human CR1 containing all 30 extra-cellular SCRdomains, is a specific example of a CR-1-like protein.

The complement receptor proteins of this invention can be made by avariety of techniques. If full length native chains are required, thenthe native molecules may be extracted from the above-identified cellsources. When soluble forms are desired, fragments of the native fulllength molecules are preferred. Accordingly, DNAs encoding the desiredchain fragments, are expressed as recombinantly produced proteinfragments. This invention is particularly useful for the purification ofsCR1 from conditioned cell culture medium of a variety of sCR1 producingrecombinant cell lines. Although one may expect some variation from cellline to cell line and among the various complement receptor products,based on the disclosure herein, it is well within the purview of one ofordinary skill in this art to adapt the invention herein to a particularcombination of complement receptor protein and producing cell line.

Generally, genes encoding proteins such as complement receptors may becloned by incorporating DNA fragments coding for the desired regions ofthe polypeptide into a recombinant DNA vehicle (e.g., vector) andtransforming or tansfecting suitable prokaryotic or eukaryotic hosts.Suitable prokaryotic hosts include but are not limited to Escherichia,Streptomyces, Bacillus and the like. Suitable eukaryotic hosts includebut are not limited to yeast, such as Saccharomyces and animal cells inculture such as VERO, HeLa, mouse C127, Chinese hamster ovary (CHO),WI-38, BHK, COS, MDCK, and insect cell lines. Particularly preferredhost are CHO cell lines deficient in dihydrofolate reductase such asATCC CRL 1793, CRL 9096 and other cell lines described hereinbelow. Suchrecombinant techniques have now become well known and are described inMethods in Enzymology, (Academic Press) Volumes 65 and 69 (1979), 100and 101 (1983), and the references cited therein. An extensive technicaldiscussion embodying most commonly used recombinant DNA methodologiescan be found in Maniatis, et al., Molecular Cloning, Cold Spring HarborLaboratory (1982) or Current Protocols in Molecular Biology, GreenePublishing (1988,1991).

One way of obtaining a DNA fragment encoding a desired polypeptide suchas a complement receptor is via cDNA cloning. In this process, messengerRNA (mRNA) is isolated from cells known or suspected of producing thedesired protein. Through a series of enzymatic reactions, the mRNApopulation of the cells is copied into a complementary DNA (cDNA). Theresulting cDNA is then inserted into cloning vehicles and subsequentlyused to transform a suitable prokaryotic or eukaryotic host. Theresulting cDNA "library" is comprised of a population of transformedhost cells, each of which contain a single gene or gene fragment. Theentire library, in theory, provides a representative sample of thecoding information present in the mRNA mixture used as the startingmaterial.

The libraries can be screened using nucleic acid or antibody probes inorder to identify specific DNA sequences. Once isolated, these DNAsequences can be modified or can be assembled into complete genes.Alternatively, as described in this invention, specific fragments of agene can be engineered independently of the rest of the gene. Proteinfragments encoded by these engineered gene fragments may not be found innature, yet they may have significant utility in the treatment ofundesirable physiological conditions. The genetic engineering of solublecomplement receptor for the prevention and/or treatment of disordersinvolving complement activity is one such case.

Once the gene or gene fragment has been cloned, the DNA may introducedinto an expression vector and that construction used to transform anappropriate host cell. An expression vector is characterized as havingexpression control sequences as defined herein, such that when a DNAsequence of interest is operably linked thereto, the vector is capableof directing the production of the product encoded by the DNA sequenceof interest in a host cell containing the vector. With specificreference to this invention, it is possible to assemble fragments of asingle coding sequence such that upon expression a soluble receptorprotein is formed. A particularly efficacious application of thisprotocol to SCR1 recombinant production is found in the Fearon, et al.PCT Applications WO89/09220, published Oct. 5, 1989, and WO91/05047published on Apr. 18, 1991, cited above.

After the recombinant product is produced it is desirable to recover theproduct. If the product is exported by the cell producing it, theproduct can be recovered directly from the cell culture medium. If theproduct is retained intracellularly, the cells must be physicallydisrupted by mechanical, chemical or biological means in order to obtainthe intracellular product.

In the case of a protein product, the purification protocol should notonly provide a protein product that is essentially free of otherproteins, by which is meant at least 80% and preferably greater than 95%pure with respect to total protein in the preparation, but alsoeliminate or reduce to acceptable levels other host cell contaminants,DNA, RNA, potential pyrogens and the like.

As mentioned above, a variety of host cells may be used for theproduction of the receptors of this invention. The choice of aparticular host cell is well within the purview of the ordinary skilledartisan taking into account, inter alia, the nature of the receptor, itsrate of synthesis, its rate of decay and the characteristics of therecombinant vector directing the expression of the receptor. The choiceof the host cell expression system dictates to a large extent the natureof the cell culture procedures to be employed. The selection of aparticular mode of production be it batch or continuous, spinner or airlift, liquid or immobilized can be made once the expression system hasbeen selected. Accordingly, fluidized bed bioreactors, hollow fiberbioreactors, roller bottle cultures, or stirred tank bioreactors, withor without cell microcarrier may variously be employed. The criteria forsuch selection are appreciated in the cell culture art. They are notdetailed herein because they are outside the scope of this invention.This invention relates to the purification of complement receptors giventheir existence in a conditioned cell culture medium.

As mentioned above this invention relates, inter alia, to application ofimmobilized metal affinity chromatography (IMAC) to the purification ofcomplement receptor proteins. The principles of IMAC are generallyappreciated. It is believed that adsorption is predicated on theformation of a metal coordination complex between a metal ion,immobilized by chelation on the adsorbent matrix, and accessibleelectron donor amino acids on the surface of the protein to be bound.The metal-ion microenvironment including, but not limited to, thematrix, the spacer arm, if any, the chelating ligand, the metal ion, theproperties of the surrounding liquid medium and the dissolved solutespecies can be manipulated by the skilled artisan to affect the desiredfractionation.

Not wishing to be bound by any particular theory as to mechanism, it isfurther believed that the more important amino acid residues in terms ofbinding are histidine, tryptophan and probably cysteine. Since one ormore of these residues are generally found in proteins, one might expectall proteins to bind to IMAC columns. However, the residues not onlyneed to be present but also accessible (e.g., oriented on the surface ofthe protein) for effective binding to occur. To that end this inventionalso contemplates the addition of appropriate residues to the complementreceptor proteins of interest. The residues, for example poly-histidinetails added to the amino terminus or carboxy terminus of the protein,can be engineered into the recombinant expression systems describedherein by following the protocols described in U.S. Pat. No. 4,569,794.

The nature of the metal and the way it is coordinated on the column canalso influence the strength and selectivity of the binding reaction.Matricies of silica gel, agarose and synthetic organic molecules such aspolyvinyl-methacrylate co-polymers can be employed. The matriciespreferably contain substituents to promote chelation. Substituents suchas iminodiacetic acid (IDA) or its tris (carboxymethyl) ethylene diamine(TED) can be used. IDA is preferred. A particularly useful IMAC materialis a polyvinyl methacrylate co-polymer substituted with IDA availablecommercially, e.g., as TOYOPEARL AF-CHELATE 650M (ToyoSoda Co.; Tokyo.The metals are preferrably divalent members of the first transitionseries through to zinc. Although Co⁺⁺, Ni⁺⁺, Cd⁺⁺ and Fe⁺⁺⁺ can be used.An important selection parameter is, of course, the affinity of theprotein to be purified for the metal. Cu⁺⁺ is preferred. Of the fourcoordination positions around these metal ions, at least one is occupiedby a water molecule which is readily replaced by a stronger electrondonor such as a histidine residue at slightly alkaline pH.

In practice the IMAC column is "charged" with metal by pulsing with aconcentrated metal salt solution followed by water or buffer. The columnoften acquires the color of the metal ion (except for zinc). Often theamount of metal is chosen so that approximately half of the column ischarged. This allows for slow leakage of the metal ion into thenon-charged area without appearing in the eluate. A pre-wash withintended elution buffers is usually carried out. Sample buffers maycontain salt up to 1M or greater to minimize nonspecific ion-exchangeeffects. Adsorption of proteins is maximal at higher pHs. Elution isnormally either by lowering of pH to protonate the donor groups on theadsorbed protein, or by the use of stronger complexing agent such asimidazole, or glycine buffers at pH 9. In these latter cases the metalmay also be displaced from the column. Linear gradient elutionprocedures can also be benefically employed.

As mentioned above IMAC is particularly useful when used in combinationwith other protein purification techniques. That is to say it ispreferred to apply IMAC to material that has been partially purified byother protein purification procedures. By the term "partially purified"is meant a protein preparation in which the protein of interest ispresent in at least 5 percent by weight, more preferably at least 10%and most preferably at least 45%. Accordingly, the application of IMACis best appreciated in the context of an overall purification protocolfor complement receptor proteins. A particularly useful combinationchromatographic protocol is disclosed in U.S. Pat. No. 5,252,216 granted12 Oct. 1993, the contents of which are incorporated herein byreference. It has been found to be useful, for example, to subject asample of conditioned cell culture medium to partial purification priorto the application of IMAC. By the term "conditioned cell culturemedium" is meant a cell culture medium which has supported cell growthand/or cell maintenance and contains secreted product. A concentratedsample of such medium is subjected to one or more protein purificationsteps prior to the application of a IMAC step. The sample may besubjected to ion exchange chromatography as a first step. As mentionedabove various anionic or cationic substituents may be attached tomatrices in order to form anionic or cationic supports forchromatography. Anionic exchange substituents includediethylaminoethyl(DEAE), quaternary aminoethyl(QAE) and quaternaryamine(Q) groups. Cationic exchange substituents include carboxymethyl(CM), sulfoethyl(SE), sulfopropyl(SP), phosphate(P) and sulfonate(S).Cellulosic ion exchange resins such as DE23, DE32, DE52, CM-23, CM-32and CM-52 are available from Whatman Ltd. Maidstone, Kent, U.K.SEPHADEX®-based and cross-linked ion exchangers are also known. Forexample, DEAE-, QAE-, CM-, and SP-dextran supports under the tradenameSEPHADEX® and DEAE-, Q-, CM-and S-agarose supports under the tradenameSEPHAROSE® are all available from Pharmacia AB. Further both DEAE and CMderivitized ethylene glycol-methacrylate copolymer such as TOYOPEARLDEAE-650S and TOYOPEARL CM-650S are available from Toso Haas Co.,Philadelphia, Pa. Because elution from ionic supports usually involvesaddition of salt and because, as mentioned previously IMAC is enhancedunder increased salt concentrations, the introduction of a IMAC stepfollowing an ionic exchange chromatographic step or other salt mediatedpurification step is particularly preferred. Additional purificationprotocols may be added including but not necessarily limited to HIC,further ionic exchange chromatography, size exclusion chromatography,viral inactivation, concentration and freeze drying.

Hydrophobic molecules in an aqueous solvent will self-associate. Thisassociation is due to hydrophobic interactions. It is now appreciatedthat macromolecules such as proteins have on their surface extensivehydrophobic patches in addition to the expected hydrophilic groups. HICis predicated, in part, on the interaction of these patches withhydrophobic ligands attached to chromatographic supports. A hydrophobicligand coupled to a matrix is variously referred to herein as an HICsupport, HIC gel or HIC column. It is further appreciated that thestrength of the interaction between the protein and the HIC support isnot only a function of the proportion of non-polar to polar surfaces onthe protein but by the distribution of the non-polar surfaces as well.

A number of matrices may be employed in the preparation of HIC columns,the most extensively used is agarose. Silica and organic polymer resinsmay be used. Useful hydrophobic ligands include but are not limited toalkyl groups having from about 2 to about 10 carbon atoms, such as abutyl, propyl, or octyl; or aryl groups such as phenyl. Conventional HICproducts for gels and columns may be obtained conmercially fromsuppliers such as Pharmacia LKB AB, Uppsala, Sweden under the productnames butyl-SEPHAROSE®, phenyl-SEPHAROSE® CL-4B, octyl-SEPHAROSE® FF andphenyl-SEPHAROSE® FF; Tosoh Corporation, Tokyo, Japan under the productnames TOYOPEARL Butyl 650, Ether-650, or Phenyl-650 (FRACTOGEL TSKButyl-650) or TSK-GEL phenyl-5PW; Miles-Yeda, Rehovot, Israel under theproduct name ALKYL-AGAROSE, wherein the alkyl group contains from 2-10carbon atoms, and J. T. Baker, Phillipsburg, N.J. under the product nameBAKERBOND WP-HI-propyl.

Ligand density is an important parameter in that it influences not onlythe strength of the interaction but the capacity of the column as well.The ligand density of the commercially available phenyl or octyl phenylgels is on the order of 40 μmoles/ml gel bed. Gel capacity is a functionof the particular protein in question as well pH, temperature and saltconcentration but generally can be expected to fall in the range of 3-20mg/ml of gel.

The choice of a particular gel can be determined by the skilled artisan.In general the strength of the interaction of the protein and the HICligand increases with the chain length of the of the alkyl ligands butligands having from about 4 to about 8 carbon atoms are suitable formost separations. A phenyl group has about the same hydrophobicity as apentyl group, although the selectivity can be quite different owing tothe possibility of pi-pi interaction with aromatic groups on theprotein.

Adsorption of the proteins to a HIC column is favored by high saltconcentrations, but the actual concentrations can vary over a wide rangedepending on the nature of the protein and the particular HIC ligandchosen. Various ions can be arranged in a so-called soluphobic seriesdepending on whether they promote hydrophobic interactions (salting-outeffects) or disrupt the structure of water (chaotropic effect) and leadto the weakening of the hydrophobic interaction. Cations are ranked interms of increasing salting out effect as Ba⁺⁺ <Ca⁺⁺ <Mg⁺⁺ <Li⁺ <Cs⁺<Na⁺ <K⁺ <Rb^(+<NH) ₄ ⁺. While anions may be ranked in terms ofincreasing chaotropic effect as PO₄ ⁻⁻⁻ <SO₄ ⁻⁻ <CH₃ COO⁻ <Cl⁻ <Br⁻ <NO₃⁻ <CIO₄ ⁻ <I⁻ <SCN⁻.

Accordingly, salts may be formulated that influence the strength of theinteraction as given by the following relationship:

    Na.sub.2 SO.sub.4 >NaCl>(NH.sub.4).sub.2 SO.sub.4 >NH.sub.4 Cl>NaBr>NASCN

In general, salt concentrations of between about 0.75 and about 2Mammonium sulfate or between about 1 and 4M NaCl are useful.

The influence of temperature on HIC separations is not simple, althoughgenerally a decrease in temperature decreases the interaction. However,any benefit that would accrue by increasing the temperature must also beweighed against adverse effects such an increase may have on theactivity of the protein.

Elution, whether stepwise or in the form of a gradient, can beaccomplished in a variety of ways: (a) by changing the saltconcentration, (b) by changing the polarity of the solvent or (c) byadding detergents. By decreasing salt concentration adsorbed proteinsare eluted in order of increasing hydrophobicity. Changes in polaritymay be affected by additions of solvents such as ethylene glycol or(iso)propanol thereby decreasing the strength of the hydrophobicinteractions. Detergents function as displacers of proteins and havebeen used primarily in connection with the purification of membraneproteins.

When the eluate resulting from HIC is subjected to further ion exchangechromatography, both anionic and cationic procedures may be employed.

As mentioned above, gel filtration chromatography affects separationbased on the size of molecules. It is in effect a form of molecularsieving. It is desirable that no interaction between the matrix andsolute occur, therefore, totally inert matrix materials are preferred.It is also desirable that the matrix be rigid and highly porous. Forlarge scale processes rigidity is most important as that parameterestablishes the overall flow rate. Traditional materials such ascrosslinked dextran or polyacrylamide matrices, commercially availableas, e.g., SEPHADEX® and BIOGEL®, respectively, were sufficiently inertand available in a range of pore sizes, however these gels wererelatively soft and not particularly well suited for large scalepurification. More recently, gels of increased rigidity have beendeveloped (e.g. SEPHACRYL®, ULTROGEL®, FRACTOGEL® and SUPEROSE®). All ofthese materials are available in particle sizes which are smaller thanthose available in traditional supports so that resolution is retainedeven at higher flow rates. Ethylene glycol-methacrylate copolymermatrices, e.g., such as the TOYOPEARL HW series matrices (Toso Haas) arepreferred.

For purposes of illustration only, this invention was applied to thepurification of a complement receptor of the soluble type. Morespecifically, to a soluble CR1 construct containing leader, LHR-A,LHR-B, LHR-C, LHR-D, SCR29, SCR30 regions up to and including the firstalanine residue of the transmembrane region; and corresponding to theCR1 encoding sequences in plasmid pBSCR1c of Fearon et al.; 1989, Int'l.Patent Publication Number WO89/09220, published Oct. 5, 1989(hereinafter "TP10HD"). The construction of a recombinant system for theproduction of TP10HD is detailed in the above mentioned PCT Applicationand summarized as follows.

CHO cells were trypsinized and plated out at 5×10⁵ per 60 mm dish andleft in the growth medium (Hams F12 nutrient medium (041-1765) with 1%stock glutamine (043-05030), 1% stock pen/strep (043-05070) and 10%bovine fetal calf serum (011-6290),Gibco, Paisley, Scotland) at 37° C.in a humidified incubator in an atmosphere of 5% CO₂ /95% air. After 21hours the cells were used for DNA transfection. An expression plasmidcontaining the sCR1 coding sequence from pBSCR1c was co-transfected withpSV2dhfr into a dhfr-requiring Chinese Hamster Ovary cell line(CHODUXBII). The transfection was carried in growth medium and employedthe calcium coprecipitation/ glycerol shock procedure as describedin:DNA Cloning, D. M. Glover ed. (Chap. 15, C. Gorman). Followingtransfection with pBSCR1c/pTCSgpt and pSV2dhfr, the cells weremaintained in growth medium for 46 hours under growth conditions (asdescribed above) prior to the selection procedure.

The selection and co-amplification procedure was carried out essentiallyas described by R. J. Kaufman, et al.(Mol. Cell. Biol. 5:1750-1759(1985)). Forty-six hours post transfection the cells were changed toselective medium MEM ALPHA (041-02571), 1% stock glutamine, 1% stockpen/strep (043-05070) and dialysed bovine fetal calf serum (220-6300AJ)(Gibco, Paisley, Scotland). The cells were maintained in the selectivemedium for 8-10 days until dhfr⁺ colonies appeared. When the colonieswere established the cells were changed into a selective mediumcontaining methotrexate, (A6770, Sigma Chem. Co., St. Louis, Mo.). Themethotrexate concentration was initially 0.02 μM and was increasedstepwise to 5 μM. During the amplification procedure aliquots of growthmedium from growing cells were assayed for TP10HD production by ELISA.Any complement receptor secreting recombinant cell line (e.g. ATCC CRL10052) may be used to supply the conditioned medium for purificationaccording to this invention, but a particular cell line certainly is notrequired.

A transfected CHO cell line capable of producing TP10HD can be culturedby a variety of cell culture techniques. For the application of thisinvention the particular method of culturing is not critical, howeverfor purposes of illustration, one method for cell culturing which may beused is a continuous perfusion process predicated on the VERAX fluidizedbed technology as embodied in U.S. Pat. Nos. 4,861,714; 4,863,856;4,978,616 and 4,997,753, the contents of which are incorporated byreference. Accordingly, transfected cells such as those described above,are scaled up in CCM-3 medium (a mixture of DMEM, Ham's F-12, bovineserum albumin and other nutrient supplements) supplemented with 10%fetal bovine serum (PBS) and 5 mM methotrexate (MTX). The cellpopulation was expanded in roller bottles until sufficient numbers ofcells were available for inoculating a bioreactor.

Prior to inoculation a S200 bioreactor underwent clean-in-place (CIP)and steam-in-place (SIP) cycles. It was then filled with CCM-3 mediumcontaining 5% FBS and charged with 450 grams of microspheres. Themicrospheres were conditioned with medium prior to inoculation. Thereactor was inoculated with cells and the operating parameters were: pH7.2., 37° C., inlet (bottom of fluidized bed) dissolved O₂ between 100and 400 torr, exit (top of fluidized bed) dissolved O₂ between 0 and 200torr. Following an initial batch phase, medium perfusion was initiated,with periodic increases in rate so as to maintain the glucoseconcentration at 1.0 g/L. This was continued until a sufficient numberof cells had accumulated in the reactor to inoculate a S2000 bioreactor.Following CIP and SIP, a S-2000 reactor was filled with CCM-3 mediumsupplemented with 5% FBS and 5 mM MTX and charged with 5000 grams ofmicrospheres. These microspheres were conditioned with medium prior toinoculation. The operating conditions in respect of temperature, reactorarrangement and dissolved O₂ are as given above. The microspheres fromthe S-200 reactor were aseptically transferred into the S-2000 reactorto initiate batch phase. When the glucose concentration fell below 1.5g/L, the growth phase was started by initiating medium perfusion (CCM-3,5% FBS and 5 mM MTX) at a rate sufficient to maintain the glucoseconcentration at 1.0 g/L. Cell growth was monitored on-line by measuringoxygen uptake and glucose consumption rates. When a sufficient number ofcells had accumulated within the reactor, the perfusion medium waschanged to CCM-3 supplemented with 1% FBS and 5 mM MTX, transitionmedium. Again this perfusion rate was modified so as to maintain aglucose concentration of 1.0 g/L. Following further growth in thetransition medium, the perfusion medium was changed once again to theproduction medium, CCM-3 supplemented with 5 mM MTX. The perfusion ratewas increased to maintain a glucose concentration of 1.0 g/L.Thereafter, either exit dissolved O₂ or recycle flow rate setpoints werelowered to maintain control over the reactor. The production phasetypically lasts for about 60 days.

Between 400 and 1600 liters of reactor permeate, stored a 4°-8° C., wereprocessed through a Millipore Prostak Microfiltration Unit. Thecell-free permeate from this operation supplied the ultrafiltrationstep. The permeate was concentrated 30-60× with a Millipore Spiral WoundSystem. Following concentration, the retentate was drained into aholding tank and the system was filled with 5-20 L of 50 mM phosphatebuffer, pH 7.5. The wash buffer was drained from the system and combinedwith the retentate. The ultrafiltration concentrate was filtered througha prefilter and a terminal 0.22 mm filter into a previously autoclavedNalgene bottle. Nominally 800 ml of concentrate are dispersed into eachbottle and stored frozen.

As mentioned previously, the particular recombinant production systemand the particular cell culturing protocol is outside the scope of thisinvention. The system and protocol discussed above are representative ofthe many options available to the skilled artisan and they are includedherein for purposes of illustration only. For example media obtainedfrom a stirred-tank bioreactor are equally suited as sources ofconditioned media for use with the present invention. The purificationprotocol which is the subject of this invention is applicable, with onlyroutine modification, to a variety of recombinant complement receptorand complement receptor-like proteins regardless of how they areproduced or cultured.

The purified complement receptor proteins obtained by practicing theprocess of this invention have the following properties: 1) greater than95% CR protein by weight; 2) stable to proteolytic degradation at 4° C.for at least three months; 3) low (<1 E.U./mg protein) endotoxin; 4) low(<1 pg/mg protein) DNA; 5) non-CR protein <5% by weight; and 6) virallyinactive.

The following example further illustrates this invention but is notoffered by way of limitation of the claims herein.

EXAMPLE I

INTRODUCTION

The procedure outlined below was developed for the isolation andpurification of soluble complement receptor-1 (sCR1) from conditionedcell culture medium concentrate. This process is designed to preparesCR1 of >95% protein purity while removing impurities derived from thehost cell, cell culture medium, or other raw materials. The recoveryprocedure consists of nine steps including cation and anion exchange,immobilized metal affinity, hydrophobic interaction and size exclusionchromatography, and two viral inactivation treatments. Each step isdescribed in detail below including materials, methods, and expectedresults. Steps 1 though 3 are carried out at 2°-8° C., and steps 6through 9 are performed at 18°-25° C. All buffers are prepared with WFIand filtered through a 10,000 MWCO filter before use. All columns aremonitored by UV absorbance at 280 nm and by conductivity, whereindicated. Columns are cleaned and equilibrated before use, and cleanedand stored in NaOH after each use.

The process is scaled to accomodate approximately 1000 L of mediumcontaining 100 G of crude sCR1, and requires 7-14 days to complete,depending on the scale of the operation.

STEP 1: MEDIA PRE-TREATMENT

While sting, the pH of 1000 L of cell-free conditioned medium is loweredto pH 5.2 by the addition of 1M acetic acid at a rate of 1-3 L/min. Thevolume of acetic acid required is approximately 3% the volume of themedium, and requires 15-30 minutes to add. The pH is monitoredcontinuously therafter. The pH adjustment produces a heavy precipitate.Clarification is achieved by microfiltration through a series of twoMillipore 30 inch Polygard-CR filters connected in tandem (0.5 micron).The sCR1 is recovered in the filtrate, and when 50-100 L of filtratehave accumulated, Step 2 is begun. This allows both the filtration andloading operations to occur simultaneously.

The acidification and filtration of the medium concentrate removes bothnon-sCR1 protein and non-proteinaceous material; and adjusts the sCR1containing filtrate to the appropriate pH for subsequent S SEPHAROSEcation exchange chromatography.

STEP 2: PHARMACIA S SEPHAROSE FAST FLOW CHROMATOGRAPHY

The pH 5.2 filtrate is loaded at a flow rate of 150 cm/hr (and sothroughout) onto a column of Pharmacia S Sepharose Fast Flow sulfonatesubstituted agaroses gel previously equilibrated with Buffer A. Thecolumn is washed at 150 cm/hr with 3-5 bed volumes of Buffer A, followedby 5-10 bed volumes of Buffer B. The sCR-1 is eluted with 3-5 bedvolumes of Buffer C. The entire elution peak is collected until theabsorbance decreases to 5% of the maximum observed absorbance. The sCR1elutes in approximately 1.5-2 bed volumes.

The column is cleaned and recycled by treating for at least 1 hr with0.5N NaOH, washing with WFI, and equilibrating with Buffer A. When notin use the column is stored in 0.01N NaOH.

The S SEPHAROSE cation exchange chromatography removes a largeproportion of cell and media derived impurities (particularly protein)and concentrates sCR1 in the Buffer C column eluate for furtherprocessing.

STEP 3: IMAC USING TOYOPEARL AF-CHELATE 650M

PART 1: CHARGING IMAC COLUMN WITH COPPER AND EQUILIBRATION OF THECHARGED COLUMN

The IDA-substituted polyvinyl methacrylate copolymer column is chargedwith copper as follows: 6-8 column volumes of 0.2% cupric sulfate ispassed over the column after flushing with 3 column volumes of WFI. Thecolumn is charged until a blue color is evident over the whole bed, andexcess copper is detected in the eluate stream. The column is thenflushed with 1-2 bed volumes of WFI, followed by 3-5 bed volumes ofBuffer C.

PART 2: LOAD, WASH, ELUTION, AND REGENERATION OF IMAC COLUMN

The S Sepharose cation exchange column eluate is loaded at a flow rateof 150 cm/hr (and so throughout) onto the IMAC column after charging andequilibration (see Step 3, Part 1). The column is washed at 150 cm/hrwith 3-5 bed volumes of Buffer C, followed by 5-10 bed volumes of BufferD. It is imperative that after flushing with Buffer D is complete, thecolumn be flushed with 3-5 volumes of Buffer C to bring the pH back to8, otherwise significant copper leaching will occur upon the applicationof Buffer E. The sCR1 is fluted with 3-5 bed volumes of Buffer E. Theentire elution peak is collected until the absorbance decreases to 5% ofthe maximum observed absorbance. The sCR-1 elutes in approximately 2 bedvolumes. The copper is removed by flushing with 5 bed volumes of 50 mMEDTA, since it is incompatible with the 0.5M NaOH sanitization. Theconcentrated copper effluent must be collected for proper disposalaccording to local codes.

The IMAC column is cleaned and recycled by treating for at least 1 hrwith 0.5N NaOH, washing with WFI, and equilibrating with Buffer C. Whennot in use the column is stored in 0.01N NaOH.

The IMAC removes cell and media derived impurities (particularly proteinand DNA).

STEP 4: VIRAL INACTIVATION WITH GUANIDINE AND ADDITION OF AMMONIUMSULFATE

PART 1: ADDITION OF GUANIDINE (PERFORMED AT 2°-8° C.)

The cold IMAC eluate is treated with guanidine by the addition ofone-half volume of cold Buffer F with constant stirring, over a periodof 10-15 minutes. When the addition of Buffer F is completed, thesolution is transferred to a second vessel by subsurface transfer, andheld for 6 minutes.

PART 2: ADDITION OF AMMONIUM SULFATE (PERFORMED AT 2°-8° C.)

The solution treated in Step 4, Part 1 is immediately diluted with anequal volume of cold Buffer G, over a 10-15 minute period, with constantstirring. The resulting solution is 1.0M in guanidine and 0.9M inammonium sulfate, and should be at 2°-8° C. before performing Step 5.

The guanidine treating affords retroviral inactivation, and the additionof ammonium sulfate prepares the solution for hydrophobic interactionchromatography using a butyl-substituted ethylene glycol-methacrylatecopolymer support, i.e., TOYOPEARL BUTYL chromatography.

STEP 5: TOYOPEARL BUTYL-650M CHROMATOGRAPHY (2°-8° C.)

The solution from Step 4 is loaded at a flow rate of 150 cm/hr onto acolumn of butyl substituted ethylene glycol-methacrylate copolymersupport, i.e., TOYOPEARL Butyl-650M previously equilibrated with BufferH. It is critical that the buffers and column are at 2°-8° C. Whenloading is completed the column is washed with 3-5 bed volumes of BufferH, and the bound sCR1 is eluted with Buffer I. The sCR-1 elutes in 1.5-3bed volumes. The column is stripped with 0.2N NaOH. The base wash elutesprotein impurities in a measurable peak, which is neutralized and heldfor assay.

The HIC column is cleaned and recycled by treating for at least 1 hrwith 0.5N NaOH, washing with WFI, and equilibrating with Buffer H. Whennot in use, the column is stored in 0.01N NaOH.

STEP 6: VIRAL INACTIVATION AT PH 11 AND DIAFILTRATION

The butyl eluate is adjusted to pH 11 by addition of 2.5M NaOH. Thesolution is immediately transferred to a second vessel by subsurfacetransfer, held at pH 11 for 16 minutes, and readjusted to pH 9.0 using2.5M HCl. The pH 11 treated solution is then continuously diafilteredagainst Buffer J in a tangential flow apparatus equipped with 30 kD MWCOlow-protein binding membranes (such as Filtron Omega series). Thediafiltration continues until 4-5 volumes have passed into the permeate,and the conductivity of the retentate is ≦2 mS/cm.

The pH 11 treating affords retroviral inactivation, and thediafiltration prepares the sCR1 solution for DEAE-derivitized ethyleneglycol-methacrylate copolymer anion exchange chromatography.

STEP 7: TOYOPEARL DEAE-650S ANION EXCHANGE CHROMATOGRAPHY

The solution from Step 6 is loaded at a flow rate of 150 cm/hr onto acolumn of DEAE-derivitized ethylene glycol-methacrylate copolymer, i.e.,TOYOPEARL, DEAE-650S, previously equilibrated with Buffer J. Afterloading, the column is washed with 3-5 bed volumes of Buffer J. Thebound sCR1 is eluted with a 5-column volume linear gradient startingfrom 100% Buffer J and extending to 100% Buffer K. The entire elutionpeak is collected until the absorbance decreases to 20% of the maximumabsorbance. Collection is then switched to a second container for thetailing end of the peak. The sCR-1 should elute in 1-2 bed volumes. Thecolumn is stripped by washing with 3 bed volumes of Buffer L.

The DEAE anion exchange column is cleaned and recycled by treating forat least 1 hr with 0.5N NaOH, washing with WFI, and equilibrating withBuffer J. When not in use the column is stored in 0.01N NaOH.

The DEAE anion exchange chromatography removes protein, DNA, andpotential viral impurities.

STEP 8: TOYOPEARL HW-55F CHROMATOGRAPHY

The DEAE anion exchange column eluate is loaded at a flow rate of 20cm/hr onto a size exclusion gel column of TOYOPEARL HW-55F previouslyequilibrated with Buffer M. The volume of the load should be ≦10% of thetotal bed volume, and the concentration of the load should be ≦5 mg/ml.Collect the entire peak until the absorbance decreases to 10% of themaximum absorbance. Collection is then switched to a second containerfor the tail of the peak. If multiple injections are required, pool thepeak fractions. The material is now ready for final concentration.

The size exclusion column is cleaned and recycled by treating for atleast 1 hr with 0.5N NaOH, washing with WFI, and equilibrating withBuffer M. When not in use the column is stored in 0.01N NaOH.

The size exclusion chromatography removes the last traces of lowmolecular weight protein impurities, and serves to exchange the sCR1into a solution containing components compatible with the finalformulation buffer, Buffer N.

STEP 9: CONCENTRATION AND FINAL FILTRATION

The TOYOPEARL HW-55F size exclusion column eluate is concentrated to 5-6mg/mL using a tangential flow ultrafiltration device appropriately sizedto the final volume expected (such as a Pharmacia MinisetteUltrafiltration unit or Millipore CUF unit) fitted with Filtron Omegaseries 30 kD or 100 kD MWCO membranes. Following concentration, thesolution is then continuously diafiltered against 5 volumes of Buffer N.The concentrated sCR-1 is filtered through a Millipore 0.2 micronMillipak filter into sterile containers.

BUFFERS

    ______________________________________                                        Buffer A                                                                              20 mM sodium phosphate, 60 mM NaCl, pH 5.2                            Buffer B                                                                              20 mM sodium phosphate, 100 mM NaCl, pH 6.0                           Buffer C                                                                              100 mM sodium phosphate, 500 mM NaCl, pH 8.0                          Buffer D                                                                              100 mM acetate, 1 M NaCl, pH 4.0                                      Buffer E                                                                              50 mM imidazole, 100 mM sodium phosphate, 500 mM                              NaCl, pH 8.0                                                          Buffer F                                                                              6 M guanidine hydrochloride, 100 mM sodium                                    phosphate, pH 7.0                                                     Buffer G                                                                              1.8 M ammonium sulfate, 100 mM sodium                                         phosphate, pH 7.0                                                     Buffer H                                                                              0.9 M ammonium sulfate, 100 mM sodium                                         phosphate, pH 7.0                                                     Buffer I                                                                              100 mM sodium phosphate, pH 7.0                                       Buffer J                                                                              50 mM Tris/Tris, HCl, pH 9.0                                          Buffer K                                                                              50 mM Tris/Tris, HCl, 0.2 M NaCl, pH 9.0                              Buffer L                                                                              50 mM Tris/Tris, HCl, 1.0 M NaCl, pH 9.0                              Buffer M                                                                              10 mM sodium phosphate, 0.9% w/v NaCl, pH 7.0                         Buffer N                                                                              16.3 mM potassium phosphate, 25 mM NaCl, 2% (w/v)                             mannitol, pH 6.9                                                      ______________________________________                                    

SOLUTIONS

    ______________________________________                                        WFI                                                                           ______________________________________                                        2.5 M sodium hydroxide                                                        0.5 M sodium hydroxide                                                        0.2 M sodium hydroxide                                                        0.01 M sodium hydroxide                                                       2.5 M hydrochloric acid                                                       1 M acetic acid                                                               0.2% (w/v) cupric sulfate, pentahydrate (CuSO.sub.4 · 5H.sub.2       O)                                                                            50 mM edetate di- or tetrasodium (Na.sub.2 EDTA or N.sub.4 EDTA               ______________________________________                                    

COLUMN PARAMETERS

    ______________________________________                                                Minimum  Maximum                                                              Length,  Flow      Load    Units for                                  Column  cm       Rate, cm/hr                                                                             Ratio   Load Ratio                                 ______________________________________                                        S Sepharose                                                                           10       150       10      . . . grams                                                                   sCR-1 per liter                                                               bed volume                                 IMAC    10       150        8      . . . grams                                                                   protein per                                                                   liter bed volume                           BUTYL   10       150        9      . . . grams                                                                   protein per                                                                   liter bed volume                           DEAE    10       150       10      . . . grams                                                                   protein per                                                                   liter bed volume                           HW-55F  45        30       <10% . . .                                                                            . . . of column                                                       & <5    volume protein                                                        mg/mL . . .                                        ______________________________________                                    

    ______________________________________                                        PURIFICATION TABLE                                                                                                      Cumula-                                                       Pro-                                                                              Total . . . tive                                         Volume   sCR.1! tein!                                                                              sCR-1 Protein                                                                             Recovery                            Step     (L)     GIL)    (G/L)                                                                              (G)   (G)   (%)                                 ______________________________________                                        Medium   905     0.05    n. d.                                                                              46.2  n. d. 100                                 SSFF Eluate                                                                            45.7    0.95    2.49 43.3  11.8  94                                  IMAC Eluate                                                                            43.4    1.00    1.82 44.1  81.9  96                                  Butyl Eluate                                                                           21.6    2.10    2.24 45.2  50.1  98                                  DEAE Eluate                                                                            10.0    4.08    3.94 40.8  39.4  89                                  HW-55F   21.7    1.68    1.65 36.4  35.8  79                                  Eluate                                                                        Purified sCR-1                                                                         6.8     5.33    5.18 36.3  35.3  79                                  ______________________________________                                         sCR-1 assayed by HPLC                                                         Protein assayed by absorbance at 280 nm (ε = 1.10 mL mg.sup.-1        cm.sup.-1)                                                               

What is claimed is:
 1. A method for purifying a complement receptorprotein from a mixture containing same comprising sequentiallycontacting said mixture with a cationic chromatographic support, metalaffinity chromatographic support, a size exclusion chromatographicsupport and selectively eluting the protein from each support.
 2. Themethod according to claim 1 wherein the receptor is selected from thegroup consisting of CR1, CR2, CR3 and CR4.
 3. The method according toclaim 2 wherein the receptor is CR1 and fragments thereof.
 4. The methodaccording to claim 3 wherein the receptor is a soluble fragment of CR1.5. The method according to claim 4 wherein the receptor is TP10HD. 6.The method according to claim 1 wherein the cationic chromatographicsupport is selected from the group consisting ofcarboxymethyl-substituted cellulose, crosslinked carboxymethyl- andsulfopropyl-substituted dextrans, carboxymethyl- andsulfonate-substituted agarose, and carboxymethyl-derivitized ethyleneglycol-methacrylate copolymers, and elution is by addition of a bufferedsalt solution.
 7. The method according to claim 6 wherein the support issulfonate-substituted agarose beads and the salt is NaCl.
 8. The methodaccording to claim 6 wherein the salt solution is 100 mM sodiumphosphate, 500 mM NaCl, pH 8.0.
 9. The method according to claim 1wherein the metal affinity support is selected from the group consistingof silica, agarose and polyvinyl-methacrylate copolymers.
 10. The methodaccording to claim 9 wherein the support is substituted and thesubstituent is selected from the group consisting of iminodiacetic acid(IDA) and tris(carboxymethyl)ethylene diamine (TED).
 11. The methodaccording to claim 10 wherein the support is a polyvinyl-methacrylatecopolymer substituted with IDA.
 12. The method according to claim 1wherein the metal affinity support is a polyvinyl-methacrylate copolymersubstituted with IDA and the complement receptor protein is selectivelyeluted with an imidazole salt buffer.
 13. The method according to claim12 wherein imidazole salt elution buffer comprises 50 mM imidazole, 100mM sodium phosphate, 500 mM NaCl, pH 8.0.
 14. The method according toclaim 1 wherein the size exclusion chromatographic support is selectedfrom the group consisting of crosslinked dextrans, polyacrylamides,agarose beads, and ethylene glycol-methacrylate copolymer matrices. 15.The method according to claim 14, wherein the support is an ethyleneglycol-methacrylate copolymer and the elution is with 10 mM sodiumphosphate, 0.9% w/v NaCl, pH 7.0.
 16. A method for the purification of acomplement receptor protein from conditioned cell culture mediumcontaining same comprising sequentially subjecting the medium to (a)cationic exchange chromatography, (b) immobilized metal affinitychromatography, (c) hydrophobic interaction chromatography, (d) anionicexchange chromatography, and (e) size exclusion chromatography.
 17. Themethod according to claim 16 wherein the cationic exchangechromatography step employs a support selected from the group consistingof carboxymethyl-cellulose, crosslinked carboxymethyl- andsulfopropyl-dextrans, carboxymethyl- and sulfonate-agarose beads, andcarboxymethyl-derivitized ethylene glycol-methacrylate copolymer, andelution is by a buffered salt solution.
 18. The method according toclaim 17 wherein the support is sulfonate-substituted agarose beads andthe salt is NaCl.
 19. The method according to claim 18 wherein the saltsolution is 100 mM sodium phosphate, 500 mM NaCl, pH 8.0.
 20. The methodaccording to claim 17 wherein the immobilized metal affinitychromatography support is selected from the group consisting of silica,agarose, and polyvinyl-methacrylate copolymers.
 21. The method accordingto claim 20 wherein the support is substituted and the substituent isselected from the group consisting of iminodiacetic acid (IDA) andtris(carboxymethyl)ethylene diamine (TED).
 22. The method according toclaim 21 wherein the support is a polyvinyl-methacrylate copolymersubstituted with IDA.
 23. The method according to claim 16 wherein theimmobilized metal affinity chromatography support is apolyvinyl-methacrylate copolymer substituted with IDA and the complementreceptor protein is selectively eluted with an imidazole salt buffer.24. The method according to claim 23 wherein imidazole salt elutionbuffer comprises 50 mM imidazole, 100 mM sodium phosphate, 500 mM NaCl,pH 8.0.
 25. The method according to claim 16 wherein the hydrophobicinteraction chromatographic support is selected from the groupconsisting of C₂ -C₁₀ alkyl-substituted agarose, aryl-substitutedagarose, alkyl-substituted silica, and alkyl-substituted organic polymerresin.
 26. The method according to claim 25 wherein the support isselected from the group consisting of butyl-, phenyl- andoctyl-substituted agarose beads and butyl-, phenyl- andether-substituted ethylene glycol-methacrylate copolymers.
 27. Themethod according to claim 26 wherein the support is a butyl-substitutedethylene glycol-methacrylate copolymer.
 28. The method according toclaim 20 wherein the support is a butyl-substituted ethyleneglycol-methacrylate copolymer and the protein is selectively eluted witha low salt buffer.
 29. The method according to claim 28 wherein theprotein is selectively eluted with a buffer containing 100 mM sodiumphosphate, pH 7.0.
 30. The method according to claim 16 wherein saidanionic exchange chromatography employs a support selected from thegroup consisting of diethylaminoethyl-substituted cellulose, crosslinkeddiethylaminoethyl-substituted dextan, quarternary aminoethyl-substituteddextrans, diethylaminoethyl-substituted agarose, quarternaryamino-substituted agarose, and diethylaminoethyl-derivitized ethyleneglycol-methacrylate copolymer.
 31. The method according to claim 30wherein said support is diethylaminoethyl-derivitized ethyleneglycol-methacrylate copolymer.
 32. The method according to claim 16wherein the size exclusion chromatography step employs a supportselected from the group consisting of crosslinked dextrans,polyacrylamides, agarose beads, and ethylene glycol-methacrylatecopolymer matrices.
 33. The method according to claim 32 wherein thesupport is an ethylene glycol-methacrylate copolymer.
 34. A method forpurifying a complement receptor protein from a conditioned cell mediumcomprising:(a) concentrating the conditioned cell medium; (b) adsorbingthe complement receptor protein onto a cationic chromatographic support;(c) washing the adsorbed protein with at least one buffer; (d) elutingthe washed protein onto an immobilized metal affinity chromatographicsupport; (e) adsorbing the eluted protein from step (d); (f) washing theadsorbed protein with at least one buffer; (g) eluting the washedprotein; (h) adsorbing the eluted protein from step (g) onto ahydrophobic interaction chromatographic support; (i) selectively elutingthe protein; (j) adsorbing the eluate from step (i) onto an anionicexchange support; (k) eluting the adsorbed protein; (l) subjecting theeluate from step (k) to size exclusion chromatography and (m) recoveringthe protein therefrom.
 35. The method according to claim 34 whichincludes the optional step or steps of inactivating viruses, if present.36. The method according to claim 35 wherein said viral inactivationstep or steps is performed after step (i) and before step (j) and/or isperformed after step (g) and before step (h).
 37. The method accordingto claim 36 wherein said viral inactivation step(s) comprises treatmentof the eluate with base or with guanidine hydrochloride.
 38. The methodaccording to claim 34 wherein the cationic exchange chromatographicsupport of step (b) is sulfonate-substituted agarose beads.
 39. Themethod according to claim 34 wherein the immobilized metal affinitychromatographic support is a polyvinyl-methacrylate copolymersubstituted with IDA.
 40. The method according to claim 34 wherein theanionic exchange chromatographic support of step (j) is selected fromthe group consisting of diethylaminoethyl, quaternary animoethyl andquaternary amine substituted resins, which resins are selected from thegroup consisting of crosslinked dextrans, agarose, and ethyleneglycol-methacrylate copolymers.
 41. The method according to claim 40wherein the anionic exchange chromatographic support is adiethylaminoethyl-substituted ethylene glycol-methacrylate copolymer.42. The method according to claim 34 wherein the hydrophobic interactionchromatographic support of step (h) is selected from the groupconsisting of C₂ -C₁₀ alkyl-substituted agarose, aryl-substitutedagarose, alkyl-substituted silica, and alkyl-substituted organic polymerresin.
 43. The method according to claim 42 wherein the support isselected from the group consisting of butyl-, phenyl- andoctyl-substituted agarose beads and butyl-, phenyl- andether-substituted ethylene glycol-methacrylate copolymers.
 44. Themethod according to claim 43 wherein the support is a butyl-substitutedethylene glycol-methacrylate copolymer.
 45. The method according to anyof claim 35 wherein the size exclusion chromatography step employs anethylene glycol-methacrylate copolymer gel.
 46. The method of claim 34wherein said protein is recovered by pooling and concentrating theprotein containing fractions from chromatography step (l) byultrafiltration.